During lymphopoiesis a pluripotent stem cell develops into T or B lymphocytes. The thymus provides a specialized environment for terminal T lymphocyte differentiation. By studying natural mouse mutants and "knock-out" mice, several of the important genes in lymphocyte development and thymic organogenesis have been recently characterized. A full understanding of lymphopoiesis would also require the identification and hierarchical ordering of very early regulatory genes. To achieve this understanding we propose to take a novel approach using the zebrafish (Danio rerio) as a genetic model system. The zebrafish combines genetic and embryologic advantages and thus offers an ideal model system to study lymphopoiesis. A mutagenesis screen was done using chemical mutagenesis and ionizing radiation. F2 progeny from mutagenized fish were screened with probes for the recombination activating gene-1 (Rag-1) and alpha embryonic globin by whole mount in-situ hybridization. Of 17 potential mutants with absent Rag-1 staining in the thymus, eight were confirmed to date by F2 incrossing. One of these mutants, CZ-3, is an autosomal recessive mutation which results in microcephaly, microphthalmia and abnormalities of the pharyngeal arches. Lymphoid markers, including the earliest gene in lymphoid ontogeny Ikaros, are totally absent in thymi from CZ-3. Histologic sections show a rudimentary thymus and complete absence of lymphocytes by ultrastructural analysis. Other hematopoietic lineages are normal in CZ-3. I have undertaken a positional cloning strategy to identify the CZ-3 gene. I have mapped the mutated gene with a closely linked marker, z10517 (0.33 cM based on 9/2744 meiotic recombinants) and have established a YAC contig distal to z10517. The zebrafish homolog of whn, the nude mouse gene, colocalizes with z10517 on the same YAC clone. whn is an attractive candidate for the gene mutated in CZ-3, as the immune deficiency in the nude mouse is the result of a rudimentary thymus. A specific aim of this proposal is to test if CZ-3 and whn are identical. If they are not, I will continue the chromosomal walk and clone CZ-3 by genetic and functional means. If CZ-3 and whn are identical I will map the remaining mutants. I will select two mutants with interesting phenotypes (which fall into two groups: those with normal and with abnormal pharyngeal arches) for fine-mapping. Cloning of these genes will further our understanding of phylogeny of the immune system as well as of molecular events in both normal T lymphopoiesis and diseases such as immune deficiencies and leukemias and will be instrumental in devising novel therapeutic strategies.